The present invention concerns a nucleic acid which codes for an autoactivated resistance protein for generating a resistance to pathogens in plants, the use of the nucleic acid for producing a transgenic plant as well as transgenic plants.
Plant diseases caused by fungi, virus, nematodes and bacteria cause large losses to harvest worldwide, compromise the quality of the harvested products and necessitate the costly and laborious application of chemical pesticides, since the natural defense mechanism of plants to fend against or delay and curb the spread the majority of potential pathogens frequently do not suffice. These defense mechanisms include hypersensitive reactions, the controlled cellular death of the host tissue at the infection site, the strengthening of the plant cell wall by lignification and callus formation, the formation of phytoalexins and the production of PR-(pathogenesis-related) proteins. The plant resistance genes (R-genes) are key molecules for the activation of the induced defense mechanisms. According to Flohr's gene-for-gene postulate the protein of an R-gene interacts with a corresponding protein of a microbial avirulence gene (Avr-gene) and thereby triggers the induced defensive reaction.
The majority of the R-genes can be categorized into five classes corresponding to the structure of the R-proteins for which they code (Martin et al, 2003). Class 1 includes only the Pto-gene of the tomato, which codes for a serin/threonine-kinase. The majority of the plant R-genes however belong to the superfamily NBS-LRR-genes, which code for a “nucleotide biding site” (NBS) and a “leucine rich repeat” (LRR). NBS-LRR-genes which exhibit on their N-terminus a “coiled-coil”-structure (CC) such as, for example, a “leucine zipper”, are categorized as CC-NBS-LRR-genes of Class 2. R-genes of CC-NBS-LRR-type are found in all angiosperms. Class 3 includes the R-genes of TIR-NBS-LRR-type, which carry on the N-terminus in place of a C-domain a sequence with homology to the animal TIR-region (“toll-interleukin-1-receptor”). Although the TIR-NBS-LRR-genes comprise approximately 75% of the R-genes in Arabidopsis thaliana, they are however not found in grasses nor in sugar beets (Tian et al., 2004).
The fourth class of the R-genes is formed by the Cf-gene of the tomato. CF-proteins have no NBS-domain, however a transmembrane domain (TM) and an extracellular LRR. The fifth class includes the Xa21-protein from rice, which is constructed from an extracellular LRR-domain, a transmembrane-domain and an intracellular kinase-domain.
While R-genes are only weakly expressed by the R-gene promoters, a strong, constitutive expression of R-genes of Classes 1, 2 and 3 results in an activation of the plant pathogen defense mechanism even in the absence of a corresponding avirulence gene product and therewith in autoactivation of the R-protein (Tang et al., 1999; Oldroyd and Staskawicz, 1998; Bendahmane et al., 2002).
Generally however the constitutive overexpression of R-genes in transgenic plants is associated with agronomically undesired characteristics, such as micronecrosis (Tang et al., 1999) or dwarfism of the plants (Frost et al., 2004).
A further possibility of the autoactivation of R-proteins of Classes 2 and 3 is the mutagenesis of special, conserved amino acid motifs in the complete CC-NBS-LRR or, as the case may be, TIR-NBS-LRR proteins. The mutagenesis of sequences in the NBS- or, as the case may be, LRR domains of the Rx-gene of the potato (Bendahmane et al., 2002) and the NBS-domains of the L6-gene of flax (Howles et al., 2005) results in mutants, which, in the absence of the corresponding avirulence gene, after transient expression, initiate cell death.
Deletion experiments with the Rx-gene show that deletion products comprised of the CC-domains and parts of the NBS-domain likewise can trigger a cell death after their transient overexpression, which occurs more rapidly than in the case of use of the full-length R-gene. These deletion products require, besides the CC-domains, also the P-loop, the kinase-2 and the complete kinase-3a of the NBS-domains. In contrast, a further shortening of the NBS-domain leads to a slower HR-triggering or initiation in comparison to the compete R-gene (Bendahmane et al. 2002).
An autoactivation of the L10-gene of flax, a R-gene of Class 3, could be achieved by formation of a shortened TIR-NBS-LRR-protein, which was comprised of TIR-domains and 34 amino acids of the restricted NBS-domain inclusive of the P-loop (Frost et al., 2004).
Although multiple methods of autoactivation of R-genes are known, until now no transgenic plants have been described in which the autoactivation of R-proteins leads to an elevated fungal resistance without simultaneously detracting from the agronomic characteristics. Attempts to stably transform two autoactivated full-length variants of the L6-gene respectively under the control of the native L6-resistance gene promoter or a fungus induced promoter in flax resulted either in normal growth fungal susceptible or to dwarf fungal resistant plants (Howels et al., 2005).